This project will explore the mechanisms that govern the assembly, sorting and transport of the vacuolar- type proton-translocating ATPase (V-ATPase) in the simple model eukaryote, the yeast Saccharomyces cerevisiae. The V-ATPase is a molecular machine that couples the hydrolysis of ATP with the translocation of protons into the lumen of organelles, and the V-ATPase is highly conserved across fungi, plants and animals. Our genetic analysis in yeast has identified a group of genes in yeast encoding proteins that function in the endoplasmic reticulum (ER) in assembly of the membrane sector of the V-ATPase. These V-ATPase assembly factors will be characterized by genetic and biochemical approaches, to characterize the assembly pathway for the V-ATPase. Whereas it has been known for years that the V-ATPase is highly conserved, it has only very recently become clear that the V-ATPase assembly factors that we have identified in yeast are conserved in humans. Three specific aims are proposed. The first aim focuses on the important question of how each of the V- ATPase assembly factors functions in the highly orchestrated assembly of the 6-subunit integral membrane domain of the V-ATPase in the ER. This aim is also focused on investigating the mechanism of ER exit of the V-ATPase, and the function of the proteins identified as required for ER exit of this protein complex. The second aim centers on characterizing the human V-ATPase assembly factors in yeast. Our collaboration with Professor Dirk Lefeber has provided us with the cDNAs of four human V-ATPase assembly factors (hVma12, hVma21, hVma22 and Ac45), as well as unpublished information on mutant alleles of these genes that cause human disease. These proteins will be characterized for V-ATPase assembly function in the much simpler yeast model system. Finally, the third aim addresses the sorting and retention of the Golgi-endosomal form of the yeast V-ATPase that contains the second isoform of subunit a, the Stv1 protein. We will use the knowledge of the Stv1 sorting/retention signal that we have recently identified to investigate and characterize the proteins involved in the sorting and retention of the Stv1-containing V-ATPase complex. The knowledge gained from these studies will provide new insights into the assembly, transport and sorting of this complex protein machine, and will provide insights into the mechanistic basis for the growing number of V-ATPase associated human diseases.